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During cytoskeleton remodeling, cancer cells generate force at the plasma membrane that originates from chemical motors (e.g., actin). This force (pN) and its time course reflect the on and off-rates of the motors. We describe the design and calibration of a force-measuring device (i.e., optical tweezers) that is used to monitor this force and its time course at the edge of a cell, with particular emphasis on the temporal resolution of the instrument.

Fluorescence microscopy is a fundamental technique for the life sciences, where biocompatible and photostable photoluminescence probes in combination with fast and sensitive imaging systems are continually transforming this field. A wide-field time-gated photoluminescence microscopy system customised for ultrasensitive imaging of unique nanoruby probes with long photoluminescence lifetime is described. The detection sensitivity derived from the long photoluminescence lifetime of the nanoruby makes it possible to discriminate signals from unwanted autofluorescence background and laser backscatter by employing a time-gated image acquisition mode. This mode enabled several-fold improvement of the photoluminescence imaging contrast of discrete nanorubies dispersed on a coverslip. It enabled recovery of the photoluminescence signal emanating from discrete nanorubies when covered by a layer of an organic fluorescent dye, which were otherwise invisible without the use of spectral filtering approaches. Time-gated imaging also facilitated high sensitivity detection of nanorubies in a biological environment of cultured cells. Finally, we monitor the binding kinetics of nanorubies to a functionalised substrate, which exemplified a real-time assay in biological fluids. 3D-pseudo colour images of nanorubies immersed in a highly fluorescent dye solution. Nanoruby photoluminescence is subdued by that of the dye in continuous excitation/imaging (left), however it can be recovered by time-gated imaging (right). At the bottom is schematic diagram of nanoruby assay in a biological fluid., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Perinatal hypoxic-ischemic brain injury results in neonatal hypoxic-ischemic encephalopathy and serious long-term neurodevelopmental sequelae. Magnetic resonance imaging (MRI) of the brain is an ideal and safe imaging modality for suspected hypoxic-ischemic injury. The pattern of injury depends on brain maturity at the time of insult, severity of hypotension, and duration of insult. Time of imaging after the insult influences the imaging findings. Mild to moderate hypoperfusion results in germinal matrix hemorrhages and periventricular leukomalacia in preterm neonates and parasagittal watershed territory infarcts in full-term neonates. Severe insult preferentially damages the deep gray matter in both term and preterm infants. However, associated frequent perirolandic injury is seen in term neonates. MRI is useful in establishing the clinical diagnosis, assessing the severity of injury, and thereby prognosticating the outcome. Familiarity with imaging spectrum and insight into factors affecting the injury will enlighten the radiologist to provide an appropriate diagnosis.

The rapid uptake of nanomaterials in life sciences calls for the development of universal, high-yield techniques for their production and interfacing with biomolecules. Top-down methods take advantage of the existing variety of bulk and thin-film solid-state materials for improved prediction and control of the resultant nanomaterial properties. We demonstrate the power of this approach using high-energy ball milling (HEBM) of alumina (Al2O3). Nanoalumina particles with a mean size of 25 nm in their most stable α-crystallographic phase were produced in gram quantities, suitable for biological and biomedical applications. Nanomaterial contamination from zirconia balls used in HEBM was reduced from 19 to 2% using a selective acid etching procedure. The biocompatibility of the milled nanomaterial was demonstrated by forming stable colloids in water and physiological buffers, corroborated by zeta potentials of +40 mV and -40 mV and characterized by in vitro cytotoxicity assays. Finally, the feasibility of a milled nanoalumina surface in anchoring a host of functional groups and biomolecules was demonstrated by the functionalization of their surface using facile silane chemistry, resulting in the decoration of the nanoparticle surface with amino groups suitable for further conjugation of biomolecules.

Innovative luminescent nanomaterials, termed upconversion nanoparticles (UCNPs), have demonstrated considerable promise as molecular probes for high-contrast optical imaging in cells and small animals. The feasibility study of optical diagnostics in humans is reported here based on experimental and theoretical modeling of optical imaging of an UCNP-labeled breast cancer lesion. UCNPs synthesized in-house were surface-capped with an amphiphilic polymer to achieve good colloidal stability in aqueous buffer solutions. The scFv4D5 mini-antibodies were grafted onto the UCNPs via a high-affinity molecular linker barstar:barnase (Bs:Bn) to allow their specific binding to the human epidermal growth factor receptor HER2/neu, which is overexpressed in human breast adenocarcinoma cells SK-BR-3. UCNP-Bs:Bn-scFv4D5 biocomplexes exhibited high-specific immobilization on the SK-BR-3 cells with the optical contrast as high as 10:1 benchmarked against a negative control cell line. Breast cancer optical diagnostics was experimentally modeled by means of epi-luminescence imaging of a monolayer of the UCNP-labeled SK-BR-3 cells buried under a breast tissue mimicking optical phantom. The experimental results were analyzed theoretically and projected to in vivo detection of early-stage breast cancer. The model predicts that the UCNP-assisted cancer detection is feasible up to 4 mm in tissue depth, showing considerable potential for diagnostic and image-guided surgery applications.

Nanoparticles have recently emerged as an important group of materials used in numerous disciplines within the life sciences, ranging from basic biophysical research to clinical therapeutics. Luminescent nanoparticles make excellent optical bioprobes significantly extending the capabilities of alternative fluorophores such as organic dyes and genetically engineered fluorescent proteins. Their advantages include excellent photostability, tunable and narrow spectra, controllable size, resilience to environmental conditions such as pH and temperature, combined with a large surface for anchoring targeting biomolecules. Some types of nanoparticles provide enhanced detection contrast due to their long emission lifetime and/or luminescence wavelength blue-shift (anti-Stokes) due to energy upconversion. This topical review focuses on four key types of luminescent nanoparticles whose emission is governed by different photophysics. We discuss the origin and characteristics of optical absorption and emission in these nanoparticles and give a brief account of synthesis and surface modification procedures. We also introduce some of their applications with opportunities for further development, which could be appreciated by the physics-trained readership.

The unique luminescent properties of new-generation synthetic nanomaterials, upconversion nanoparticles (UCNPs), enabled high-contrast optical biomedical imaging by suppressing the crowded background of biological tissue autofluorescence and evading high tissue absorption. This raised high expectations on the UCNP utilities for intracellular and deep tissue imaging, such as whole animal imaging. At the same time, the critical nonlinear dependence of the UCNP luminescence on the excitation intensity results in dramatic signal reduction at (∼1 cm) depth in biological tissue. Here, we report on the experimental and theoretical investigation of this trade-off aiming at the identification of optimal application niches of UCNPs e.g. biological liquids and subsurface tissue layers. As an example of such applications, we report on single UCNP imaging through a layer of hemolyzed blood. To extend this result towards in vivo applications, we quantified the optical properties of single UCNPs and theoretically analyzed the prospects of single-particle detectability in live scattering and absorbing bio-tissue using a human skin model. The model predicts that a single 70-nm UCNP would be detectable at skin depths up to 400 µm, unlike a hardly detectable single fluorescent (fluorescein) dye molecule. UCNP-assisted imaging in the ballistic regime thus allows for excellent applications niches, where high sensitivity is the key requirement.

High-affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (K(D)) of the molecular pair, with avidin:biotin being the state-of-the-art molecular pair (K(D) ∼ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high-affinity protein pair, barstar:barnase (K(D) ∼ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non-negligible background due to the non-specific binding. A quantitative assessment of the non-specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin-based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non-specific binding, showing good prospects for high-sensitivity rare biomolecular event nanoproteomic assays., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Metamaterials, artificial electromagnetic media realized by subwavelength nano-structuring, have become a paradigm for engineering electromagnetic space, allowing for independent control of both electric and magnetic responses of the material. Whereas most metamaterials studied so far are limited to passive structures, the need for active metamaterials is rapidly growing. However, the fundamental question on how the energy of emitters is distributed between both (electric and magnetic) interaction channels of the metamaterial still remains open. Here we study simultaneous spontaneous emission of quantum dots into both of these channels and define the control parameters for tailoring the quantum-dot coupling to metamaterials. By superimposing two orthogonal modes of equal strength at the wavelength of quantum-dot photoluminescence, we demonstrate a sharp difference in their interaction with the magnetic and electric metamaterial modes. Our observations reveal the importance of mode engineering for spontaneous emission control in metamaterials, paving a way towards loss-compensated metamaterials and metamaterial nanolasers.

Aim: The author's group report, for the first time, on the development of a quantum dot (QD)-based fluorescent somatostatin (somatotropin release-inhibiting factor [SRIF]) probe that enables specific targeting of somatostatin receptors. Receptor-mediated endocytosis of SRIF was imaged using this probe., Materials & Methods: Biotinylated SRIF-analog (SRIF-B) and streptavidin (Sav)-coated QDs were used for the probe synthesis. A dye-labeled streptavidin complex was used to evaluate the effect of Sav binding on the activity of SRIF-B., Results: A preconjugated probe of the form SRIF-B:Sav-QD, was inactive and unable to undergo receptor-mediated endocytosis. An alternative in situ bioconjugation strategy, where SRIF-B and Sav-QD were added in two consecutive steps, enabled visualization of the receptor-mediated endocytosis. The process of Sav binding appeared to be responsible for the inactivity in the first case., Conclusion: The in situ two-step bioconjugation strategy allowed QDs to be targeted to somatostatin receptors. This strategy should enable flexible fluorescent tagging of SRIF for the investigation of molecular trafficking in cells and targeted delivery in live animals.

We investigate the intracellular uptake of 7.3 nm, 21.2 nm and 31.3 nm average size pure colloidal gold nanoparticles synthesized using femtosecond laser ablation technique in pure water. Dark-field imaging, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) was used to assess the uptake of these pure gold nanoparticles in the pancreatic tumor cell line. We show that these ligand-free gold nanoparticles are non-toxic to these cells. The nanoparticles and cell images indicated that unmodified gold nanoparticles interacted with the cells, despite negative surface charge on both the cells and the nanoparticles. We also demonstrate that the uptake of the gold nanoparticles is size-dependent., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Somatostatin (SST) is a peptide neurotransmitter/hormone found in several mammalian tissue types. Apart from its natural importance, labeled SST/analogues are utilized in clinical applications such as targeting/diagnosis of neuroendocrine tumors. We report on the development and characterization of a novel, recombinant, fluorescent somatostatin analogue that has potential to elucidate somatostatin-activated cell signaling. SST was genetically fused with a monomeric-red fluorescent protein (mRFP) as the fluorescent label. The attachment of SST to mRFP had no detectable effect on its fluorescent properties. This analogue's potency to activate the endogenous and transfected somatostatin receptors was characterized using assays of membrane potential and Ca(2+) mobilization and immunocytochemistry. SST-mRFP was found to be an effective somatostatin receptor agonist, able to trigger the membrane hyperpolarization, mobilization of the intracellular Ca(2+) and receptor-ligand internalization in cells expressing somatostatin receptors. This complex represents a novel optical reporter due to its red emission spectral band suitable for in vivo imaging and tracking of the somatostatin receptor signaling pathways, affording higher resolution and sensitivity than those of the state-of-the-art radiolabeling bioassays.

The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-β (Aβ) peptide. However, the monomeric state of this aggregation-prone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ± 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both Aβ(40) and Aβ(42) remain predominantly monomeric up to 3 μm, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If Aβ oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers.

Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining its resolution. We demonstrate a 4 × 4 × 18 cm(3) sized fluorescence correlation spectrometer, which requires no alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by the large background fluorescence of the commonly available optical fibers. We propose this as a portable and field deployable single molecule device with practical diagnostic applications.

We report a systematic empirical study of nanoparticle internalization into cells via non-specific pathways. The nanoparticles were comprised of commercial quantum dots (QDs) that were highly visible under a fluorescence confocal microscope. Surface-modified QDs with basic biologically significant moieties, e.g. carboxyl, amino, and streptavidin, were used, in combination with surface derivatization with polyethylene glycol (PEG) for a range of immortalized cell lines. Internalization rates were derived from image analysis and a detailed discussion about the effect of nanoparticle size, charge and surface groups is presented. We find that PEG derivatization dramatically suppresses the non-specific uptake while PEG-free carboxyl and amine functional groups promote QD internalization. These uptake variations displayed a remarkable consistency across different cell types. The reported results are important for experiments concerned with cellular uptake of surface-functionalized nanomaterials, both when non-specific internalization is undesirable and when it is intended for material to be internalized as efficiently as possible.